The hydrogels were loaded with urea, which is an important fertilizer in the field of agriculture. The PVA/GA-CH hydrogel microspheres showed a maximum loading percentage (Lmax%) of 89% (w/w), while the CS/GA-PH hydrogel microspheres showed an Lmax% = 79.75% (w/w) for urea. The urea-release behaviors of the hydrogel microspheres were studied under different release media and temperature conditions. In practice, the PVA/GA-CH hydrogel microspheres showed a better release profile in the RW at 10 °C, while the CS/GA-PH hydrogel microspheres showed a more controlled release in media at a pH 9 and at 30 °C. The urea-loaded microspheres, aside from those following the release, were characterized via FTIR and SEM. In contrast, virgin microspheres were characterized using XRD,1H NMR, (TGA and DSC), and the maximum degree of swelling, in addition to being subjected to SEM and FTIR analyses.Agricultural technical development relies exclusively on the effective delivery of agrochemicals and water to plants and on reducing the harmful effects of agrochemicals on useful organisms in the soil. In this study, super-absorbent hydrogels were prepared in the form of microspheres using gum Arabic (GA), which was copolymerized once with chitosan (CS) and once with poly  (PVA). To impart mechanical strength to the hydrogel microspheres, a covalent cross-linker ) was used for the PVA/GA hydrogel, and an ionic cross-linker (sodium hexametaphosphate (SHMP)) was used for the CS/GA hydrogel. The prepared PVA/GA-CH and CS/GA-PH hydrogel microspheres showed different degrees of swelling (DSs) in the following solution media: deionized water (DW), river water (RW), and buffered solutions . The PVA/GA-CH hydrogel microspheres showed a maximum DS of 84 g/g in the RW, while the CS/GA-PH hydrogel microspheres showed a maximum DS of 63 g/g in the buffered solution at a pH 9. The water-retention capabilities of the hydrogels were studied using a mixture of 0.5% ( Super-absorbent polymers (SAPs) are macromolecules with long chains that have soft network structures and a high ability to absorb a significant amount of water due to their three-dimensional structures .Recently, many studies have utilized hydrogels, which have received a significant amount of attention in agricultural applications, as water-management polymers that reduce the consumption of irrigation water and control the release of different fertilizers into the soil ,3. HydroHydrogels made from synthetic polymers possess many tunable properties, but they sometimes have limitations; therefore, they are modified chemically with natural polymers in order to obtain precisely controlled micro-environments and bioactive features that originate from natural polymers . Therefo1H NMR, FTIR (TGA and DSC), XRD, and SEM analyses were used. The degrees of swelling of the hydrogel microspheres were studied, and they were then loaded with urea and finally allowed to release the urea in different media solutions and at different temperatures.In this study, different hydrogels were prepared via the copolymerization of natural polymers with a synthetic polymer. The goals of this study were to prepare hydrogel microspheres for the sustained release of agrochemicals into agricultural soil and to confirm their suitable loading and releasing conditions. The other important goal was to examine the tendency of the hydrogel microspheres to absorb water and to study their water retention over a long period of time before it is released into soil for the irrigation of plants. The gum Arabic was copolymerized with chitosan and poly , and the prepared hydrogels were characterized for their structural, thermal, crystalline, and morphological properties; their characterization is considered very important for the safety of the hydrogel particles, especially as they will be used in agricultural lands. Therefore, w/w) acetic acid for purification and then filtered under pressure to remove undissolved particles of chitosan. The clear filtrate was subsequently precipitated using a 1M NaOH solution and filtered and dried at 25 °C under a vacuum. The compounds N,N′-methylenebisacrylamide (MBA), sodium hexametaphosphate (SHMP), ammonium persulphate (APS), and the urea fertilizer model were obtained from BDH, Brighouse, UK. Phosphate buffered solution pH 4 and boric buffered solution pH 9 and other chemicals were of analytical grade reagents and received from Fluka, Geneva, Switzerland. River water (RW) of a hardness of 250 ppm was collected from the Tigris river in Mosul.The poly (PVA) was obtained from Fisher Scientific, Waltham, MA, USA. Highly viscose chitosan (CS), and gum Arabic (GA) were obtained from Sigma-Aldrich . The chitosan (70% DDA) was dissolved in 2% (v/v) acetic acid in distilled water. The gum Arabic solution was prepared from 1.0 g of GA in 100 mL of distilled water. A volume of 10.0 mL of GA solution, 1.0% (w/v), was heated in a 250 mL three-necked round-bottom flask at 65 °C, using a mantle with a magnetic stirrer. The flask was connected to a nitrogen line passed through an alkaline pyrogallol solution that was free of oxygen. A volume of 5.0 mL of 10.0% (w/v) APS initiator solution was added, followed by 10.0 mL of a 1.0% (w/v) CS solution that was added stepwise using a fine-needle syringe, and the temperature of the mixed solution was kept at 65 °C with continuous, quiet stirring. Then, 5.0 ml of 2% (w/v) of an MBA covalent cross-linker was added with continuous stirring. Finally, 5.0 min later, accompanied by stirring for one hour without heating, the formed CS/GA-CH hydrogel microspheres were filtered, washed many times with distilled water, and finally dried in a vacuum oven at 30 °C.The chitosan solution was prepared from 1.0 g of CS in 100 mL of 2% (w/v) GA solution. Where, 10.0 mL of 1.0% (w/v) was heated in 250 mL three-necked round bottom flask at 65 °C using mantle with magnetic stirrer. The flask was connected to a nitrogen line passed through an alkaline pyrogallol solution that was free of oxygen. A volume of 5.0 mL of 10.0% (w/v) APS initiator solution was added. Then, 20.0 mL of the 1.0% (w/v) PVA solution was added stepwise, using a fine-needle syringe, and the temperature of the mixed solution was kept at 65 °C with continuous, quiet stirring. A volume of 5.0 mL of 6% (w/v) SHMP, the ionic cross-linker, was also added stepwise and maintained at 65 °C with stirring. Finally, the formed PVA/GA-PH hydrogel microspheres were kept in a water bath at 80 °C for 24 h before filtration.Whereas, the (PVA-co-GA) hydrogel microspheres were prepared using previous stock solution of 1.0% (t is the weight of the wet sample at a time (t), and W0 is the weight of the dry sample.A mass of 100 mg of the dry hydrogel microspheres was immersed in 20 mL of one of the following solutions (distilled water (DW), river water (RW), buffered solution ). The hydrogel was left in the swelling solution for 24 h in order to reach its maximum degree of swelling. The swelling solutions were kept at temperatures of 10 °C, 25 °C, and 50 °C for comparison. After 6 h, the hydrogel microspheres were removed, filtered with a 100-mesh sieve, and left for 10 min inside the sieve for the solution to drain. The swollen hydrogel microspheres were weighted and then returned to the solution. This process was repeated four times after 6 h, and the degree of swelling was calculated using the following equation :Degree ot represents the sample weight after the defined time, and W0 is the initial weight.A mass of 200 g of a dry agricultural soil sample was mixed with 0.1 g of the prepared hydrogel microspheres in a ventilated paper cup. Thereafter, 100 mL of river water (RW), representing the minimum rain fall level recorded in the most arid and semi-arid regions , was addmax 278 nm. The calibration curve was then used to calculate the unknown concentrations of the urea loaded in the hydrogel microspheres. Moreover, 100 mg of the hydrogel microspheres was kept in a 0.1% (w/v) solution of 100 mg of urea in 100 mL of distilled water. The loading temperature was fixed at 25 °C, room temperature. The loading time was varied between 6, 12, and 24 h in order to record the maximum loading percentage. The following equations were used for calculating the maximum loading percentage (Lmax%) and efficient loading percentage (EL%) [The concentrations of the urea loaded on the hydrogel microspheres were measured using a UV-visible JASCOV-630 spectrophotometer, Tokyo, Japan. The calibration curve was determined from different known concentrations of urea, and the microspheres’ absorbances were measured at λge (EL%) :(3)Lmax%max 278 nm and for a fixed time interval of 3 h by taking 3 mL of solution from release media and replaced with 3 mL of the native solvent. The calibration curve was used to calculate the different unknown release concentrations of urea from their absorption values. The release process was continued until no urea was released. The release of urea was given as the controlled release percentage (CRmax%) using Equation (5) [Loaded hydrogel microspheres 100 mg was allowed to release in 20 mL solution of DW, RW and buffered solution pH 9. For comparison, the release media were examined at temperatures of 10, 25, and 50 °C. The absorbance of the released urea from the hydrogel microspheres in different release media solutions were measured by UV-Visible spectrophotometer at λtion (5) :(5)Contrt is the cumulative amount of urea released at a time (t) and Using Equation (7), the overall release was calculated as the cumulative release percentage (Rcum%) for a fixed time interval of 3 h:Paying attention to agricultural soil in terms of providing water for irrigation and important nutritional elements for plants like fertilizers is considered one of the important basics in agricultural science. Hydrogels have the capacity to achieve many important and necessary applications, including agricultural uses. The goal of this study was to prepare the following PVA-co-GA/CH and CS-co-GA/PH hydrogels and mix them in low percentages with agricultural soil to examine their capabilities to retain water for long periods of time, in addition to loading them with urea, a common fertilizer, and releasing it to the plants.−1 is representative of the hydroxyl groups of both PVA and GA.The FTIR spectrum a,b shows−1 belongs to the amide group of the cross-linker MBA. The absorption frequency at 1649 cm−1 belongs to the carbonyl groups of the GA, and the bands at 2941 cm−1 and 2844 cm−1 represent the methylene groups present in the PVA, GA, and MBA. Finally, the absorption frequency at 1060 cm−1 belongs to the (-C-O-C-) of the glucopyranose units of the GA, and those at 1084 cm−1 and 1426 cm−1 belong to the (-C-O-) and (-C-OH), respectively, of both the PVA and GA.The band at 1538 cm−1 and 1531 cm−1, which represent the carbonyl groups (amide-I) and the amine group (amide-II), respectively, and absorption frequencies at 1239 cm−1, 1061 cm−1, and 740 cm−1, which belong to the υ(P-O-P) of the ionic cross-linker SHMP.The CS/GA-PH hydrogel shows, via FTIR, functional groups  similar 1H NMR of PVA/GA-CH hydrogel (hydrogel  have shomax) at which the hydrogel degraded, while the crystalline decomposition temperature (Tcr) at 800 °C has shown 83.3% weight loss where the hydrogel sample was melt.The TGA and DSC of the PVA/GA-CH and CS/GA-PH hydrogels were studied , and themax and Tcr) of CS/GA-PH hydrogel  of +488.2 and +579.8 J/g at 184.7 °C and 482.6 °C, respectively, it indicates that the PVA/GA-CH hydrogel already has a crystalline structure with a stable composition due to the presence of the PVA polymer in its structure. Therefore, it needs +488.2 and +579.8 J/g for decomposition. Even the CS/GA-PH hydrogel shows a DSC thermogram  of composites comprising a mixture of agricultural soil with either the PVA/GA-CH hydrogel or the CS/GA-PH hydrogel were studied based on Equation (2). The soil/PVA/GA-CH hydrogel microsphere composite showed the retention of water inside the soil for 42 days; moreover, it maintained the soil’s moisture for the same period . In contmax%) of urea loaded onto the PVA/GA-CH hydrogel microspheres yielded 89% after 12 h, meaning that the hydrogel microspheres reached their maximum degree of swelling after 12 h; the microspheres were able to achieve a high loading percentage. The high loading percentage (Lmax%) was due to the irregular morphology of the PVA/GA-CH hydrogel surface and the corrugates and folds present in the surface beside the holes  of the CS/GA-PH hydrogel compared with the Lmax% of the PVA/GA-CH microspheres was also because of their low DS value. However, the cationic nature of chitosan [In contrast, after 12 h, the Lchitosan  could at−1 represent the –N-H bonds of the urea. In contrast, the absorption band at 3251 cm−1 belongs to the –OH bonds of the PVA and GA polysaccharides. The bands at 1450 and 1080 cm−1 belong to the C-N bond of urea. The bands at 1020, 1385, and 826 cm−1 belong to the loaded urea.The FTIR characteristic frequencies of the important functional groups of the urea-loaded PVA/GA-CH and CS/GA-PH hydrogels are listed in −1, which represent the -P=O phosphate functional group of the SHMP in the hydrogel.On the other hand, the FTIR spectrum of the CS/GA-PH hydrogel  shows abThe SEM images of the loaded hydrogels show the urea particles interspersed between the folds of the hydrogels. The SEM image of the PVA/GA-CH hydrogel a after lAt the same time, the SEM image of the CS/GA-PH hydrogel after loading b shows tmax 278 nm and for a fixed time interval of 3 h. Accordingly, Equation (7) was used to calculate the cumulative release (Rcum%). Experimentally, it was noted that the most suitable release medium for the PVA/CS-CH hydrogel microspheres was the river water (RW) solution at 10 °C  because of its ionic bonds, which are more stable than covalent bonds. The DSC analysis showed that the PVA/GA-CH hydrogel demonstrates endothermic heat fusion, whereas the CS/GA-PH hydrogel has one exothermic heat fusion curve that is due to the decomposition of the non-crystalline structure of the chitosan. The XRD pattern of PVA/GA-CH hydrogel it shows crystalline structure return to the PVA symmetry structure and the three dimensional structure done by the covalent cross-linker, while CS/GA-PH hydrogel has less crystalline maxima done by its ionic crosslinking. The SEM images of both hydrogels showed homogeneous composites with holes and folds in their surface morphologies.Both the PVA/GA-CH and CS/GA-PH hydrogels were characterized, and their structures were examined. FTIR spectroscopy showed that the hydrogels’ functional groups appeared at their characteristic absorption frequencies. The maximum degree of swelling of the hydrogel microspheres showed a great variation depending on the composite, the electronic interactions between their chains, and the swelling media solution and its temperature. Therefore, the PVA/GA-CH hydrogel yielded a DS = 84 g/g in RW at 10 °C, while the CS/GA-PH hydrogel had a DS = 63 g/g at a pH 9 at 30 °C. The electronic interactions between the hydrogel composite, in addition to its cross-linker and the ions of the swelling media, fixed the suitable conditions for each hydrogel to reach its maximum DS.max% of the PVA/GA-CH hydrogel microspheres with urea was 89%, and this high percentage was due to the irregular morphology of the hydrogel surface and the presence of corrugates and folds in its surface, which could increase its efficiency of loading. In comparison, the Lmax% of the CS/GA-PH hydrogel with urea was 79.75%; this is still high because of the cationic nature of chitosan, which could attract the urea molecules and allow them to reach the cores of the hydrogel microspheres more easily. It was shown that the most suitable release medium for the PVA/CS-CH hydrogel microspheres was the river water (RW) solution, and the loaded urea was released in bursts for 5 h and then under controlled release for 20 h at 10 °C; this was according to the swelling conditions. In comparison, the CS/GA-PH hydrogel microspheres showed that their best release medium had a pH 9 and a temperature of 30 °C, and this was also according to the swelling conditions. Finally, the release curve of the CS/GA-PH hydrogel demonstrated more controlled behavior in comparison with the release curve of the PVA/GA-CH hydrogel because in a solution at a pH 9, the ionic hydrogel chains could control the electronic interactions and swelling to a limit that allowed the polymer to control its release more. Accordingly, the cationic functional groups of chitosan beside the anions of the cross-linker (SHMP) create a balance inside the hydrogel core and ultimately control the release of the anionic urea. All the characteristic analyses of the hydrogel microspheres show that they can be reused in drug delivery systems several times.Both hydrogels showed high levels of water retention in agricultural soil: the soil/PVA/GA-CH composite demonstrated water retention for 42 days, whereas the soil/CS/GA-PH composite retained water for 38 days; this proved that the degree of swelling of the hydrogels plays a significant role in water retention inside the soil. The L


Nano-additives results in the formation of nano-cementation (NC). This process is recently used to improve the durability of various building materials. NC used to improve the strength of untreated soil materials, also known as nano soil-improvement (NSI). In few years, the role of nano-additives in various types of soils were developed. In this research, the role of micro- and nano- size of bentonite as soil stabilizer was evaluated as first few research to improve geotechnical properties of soils. Nano-additives prepared by micro- and nano- sizes of bentonite were blend with four formulations. These formulations of micro- and nano- additives at concentrations of 0, 1, 2, and 3%, namely 0% Micro-Bentonite, 1% Micro-Bentonite, 2% Micro-Bentonite, 3% Micro-Bentonite, 0% Nano-Bentonite, 1% Nano-Bentonite, 2% Nano-Bentonite, and 3% Nano-Bentonite, respectively. These formulations of micro- and nano- additives were separately added to soil. Specimens with 3% nano-bentonite showed significant improvement in unconfined compressive strength (UCS) of soil that was more than 2.3-times higher than control specimen in 7-d curing time. Also the performance of micro-bentonite resulted in improvement in UCS of soil that was more than 1.1-times higher than control specimen at 7-d curing time. The secant modulus at 50% of peak stress (E50) of the samples treated with micro- and nano- additives increased in comparison to untreated specimens. Further, X-ray fluorescence (XRF), scanning electron microscopy, and X-ray diffraction analyses characterized micro- and nano- structures of soil specimens, and showed the performance of nano-additives in improving strength of soils. Results show that nano-bentonite as a type of nano-additives is an effective means of increasing the strength of soils. This research shows the significant of nano-bentonite in soil improvement, as a NSI technique. Using additives can be an effective way to improve soils and make them more stable. Additives can include traditional stabilizers like lime, cement, fly-ash, and related additives. Each of these materials has its own advantages and disadvantages, and it is important to study the differences between them to understand the best options for improving untreated soils1.Untreated soils are types of loose, loosely compacted soils that can be unstable and can cause problems for building foundations. Construction on untreated soils can lead to serious consequences, such as cracks in building structures and foundations. Common techniques for improving soils include deep soil mixing, stone columns, jet grouting, pre-loading and related methods, but these techniques may not be sufficient for some types of soils25. Bentonite is one of recent environment friendly additives used to improve the geotechnical properties of soils although strength is limited. Nano-additive (NA) is one of the recent methods used to improve the geotechnical properties of soils. NA is a type of nano soil-improvement (NSI) techniques. Recently, nano-additives (NA) with outstanding mechanical properties has been used for soil stabilization in a few studies25. He et al.2 evaluated the role of modified Na-bentonites in calcareous purple soil. Yu et al.3 investigated two types of organic bentonites with heavy metals in soil stabilization. Liu et al.4 tested chitosan/bentonite and Cr(VI) composites in soil improvement. Pokharel and Siddiqua5 conducted a Canadian case study on the combination of calcium bentonite clay and fly ash in organic soils. A few researchers such as Sun et al.6 evaluated the role of cadmium (Cd) and lead (Pb) using bentonite in soil improvement: Bani Baker et al.7, Liu8, Cheng et al.9 and Taha and Taha10 developed the role of nano-bentonite in sandy and clayey soils. Kozlov et al.11 evaluated the environmental sustainability performance of micro-bentonite. The composition of cement and bentonite was controlled by Li and Zhang12, Bellil et al.13, Estabragh et al.14, and Consoli et al.19. Hussein and Ali15 did a research on the role of polypropylene fiber in expansive soils while Muhammad and Siddiqua16 analyzed the feedback of bentonite magnesium-alkalinization at the micro-level in silty sand. El Aal et al. evaluated the role of sodium chloride as a case study in alluvial soils26. Muthukkumaran and Selvan17 analyzed the combination of montmorillonite-rich bentonite in clay soils. Estabragh et al.18 evaluated clay soil with MTBE as a ground improvement method. Falamaki et al.20 tested the composition of bentonite and phosphate in clay improvement. The role of microbial performance with bentonite in coarse ground improvement was investigated by Zhao et al.21 and Li et al.27. A few researchers such as Shourijeh et al.22, Firoozfar and Khosroshahiri23, and Cheng et al.9 used different micro- and nano-sized clay types in erosion, landfill, and consolidation improvement. The importance of nano-additives in soil stabilization was described in existing research25. Sakr et al.28 evaluated the role of rice husk powder as a case study in swelling soil.According to these conditions, researchers are finding new techniques in soil improvement. There are few studies where bentonite showed its potential in increasing the strength of soils28. The lack of research on the effects of the powder base of micro- and nano- additives on soils led this study to examine the effect of soil stabilization by micro-bentonite and nano-bentonite using the suspension method. Bentonite is a natural clay which is widely used in many fields, particularly in building and public works. It is used as an adjuvant in the manufacture of concrete, as a binder for soils, as a waterproofing agent for foundations, and as a containment material for nuclear waste. The advantages of bentonite include its abundant availability, low cost, ability to retain water, and ability to improve the physical and chemical properties of materials to which it is added. This study was conducted to stabilize and improve the soil properties, considering the economic justification and optimal use of the bentonite micro- and nano- particles, which had not been done in clayey sand soil previously. No previous research has prepared nanoparticles without chemicals using low-cost and energy-efficient mechanical methods, according to a literature review. This research stabilized and improved the soil properties with soluble nanoparticles, considering the economic justification and optimal use of bentonite nanoparticles. This has not been done before, to the best of our knowledge. Comparing the effects of micro-bentonite and nano-bentonite is an important research topic to understand the potential advantages of nano-bentonite over micro-bentonite. Nano-bentonite may have unique properties that may make it more effective at improving clayey sand soils than micro-bentonite but understanding the differences between the two is important to determining the best options for construction projects. This research aims to study the differences between micro-bentonite and nano-bentonite to improve clayey sand soils. The main objective of this research is to compare the effects of micro-bentonite and nano-bentonite as additives on clayey sand soils. This includes the study of the physical and mechanical properties of improved clayey sand soils, as well as their ability to withstand loads and maintain their stability over time. Tests were carried out in the laboratory to understand the differences between micro-bentonite and nano-bentonite, on clayey sand soils.According to a literature review, no research has been conducted on the preparation of nano-additives without the contribution of chemicals using lower cost and energy by mechanical methods. The most studies have been used powder particles in soil stabilizationThus, in this research, a fixed amount of micro-bentonite and nano-bentonite added from lower to high concentrations at 0, 1, 2, and 3% was used as stabilizer to improve the geotechnical properties of clayey sand soils. All such clayey sand soils were evaluated to obtain Atterberg limits, elastic modules (E50), and unconfined compressive strength (UCS). All specimens were characterized to evaluate potential of micro- and nano- bentonite in the enhancement of performance characteristics of clayey sand soils using SEM, XRD, and XRD analyses. The results of this research could be used to develop new techniques to improve untreated soils and to inform building standards and codes.Clayey sand soil samples were prepared from Survajin Aghigh in Qazvin Province, Iran. The sample soil was classified as clayey sand (SC) according to the unified soil classification system (USCS). The gradation curve, shown in Fig. The micro-bentonite was used to produce nano-bentonite. The chemical composition of nano-bentonite is given in Table The sample of clayey sand was dried in oven at 105 °C. Grain-size distribution plot for the clayey sand is given in Fig. 3 dry unit weight at 7.5% water content. The optimum water content of untreated clayey sand is 7.5%, which corresponds to the maximum dry density of 2.02 g/cm3.The standard compaction test on the graph in Fig. The Atterberg limits which determine the plasticity properties of a soil which are important for the valuation of the soil stability. The liquid limit, plastic limit, and plasticity index were determined. These values from clayey sand specimens with micro- and nano- bentonite additives were evaluated according to ASTM D 4318.A series of test specimens, each 50 mm in diameter by 100 mm in height, was prepared at optimum moisture content for UCS determinations at various curing times. These strength values for clayey sand specimens with micro- and nano- bentonite additives were evaluated based on ASTM D 2166. An axial strain rate of 1.27 mm/min was applied. These samples were tested for curing periods of 1d, 7d, and 28d. The secant modulus at 50% of peak stress (E50) of treated micro- and nano- samples was determined and compared to untreated specimen based on stress—strain behavior registered in UCS test.X-Ray Fluorescence (XRF) analysis is an analytical technique that uses X-rays to excite atoms in a sample and measure the light emitted by the excited atoms. This light can be analyzed to determine the chemical composition of the sample. X-ray fluorescence analysis is a fast and accurate way to determine the composition of many types of materials, such as minerals, metal alloys, chemicals and consumer products. It is widely used in the mining, petroleum and metallurgical industries, as well as in environmental and occupational health and safety applications. These values from clayey sand specimens with micro- and nano- bentonites were evaluated based on ASTM E1621-13 in XRF. X-ray diffraction (XRD) testing is a method used to study the crystal structure of a substance. It involves the use of X-rays to produce light diffraction on atoms in a crystal, producing an image that can be analyzed to determine the spatial arrangement of atoms in the crystal structure. The X-ray diffraction test made it possible to determine the crystal structure of bentonite. XRD was evaluated to identify minerals and other crystal structure of soil samples including treated and untreated specimens with micro- and nano- bentonites using X-ray diffractometer in Tehran, Iran. These values from clayey sand soil with micro- and nano- bentonite additives were evaluated based on BS EN 13925-1 in XRD. Scanning Electron Microscopy (SEM) analysis is an imaging technique used to study the structure and composition of materials at the micro and nano scales. It permits using an electron beam to scan the surface of a sample and produce a two-dimensional image of its surface. The microstructures of all gold-coat specimens were evaluated under a Scanning Electron Microscopy (SEM) in Tehran, Iran. These values from clayey sand specimens with micro- and nano- bentonite addives were evaluated based on 5 µ and 500 nm scales in SEM.Generally the behavior of untreated and treated clayey sand in related to the water content. According to Figs. The results of Fig. In treated soil the plasticity index increases with additive content Figs.  and 7. WThe results on the graph in Fig. 2 for an axial strain of 0.18%. The treated clayey sandy specimen with the addition of 1% micro-bentonite at 1d of maturation reaches its maximum optimum at an axial stress of 2.8 kg/cm2 for an axial strain of 0.11%. Adding 1% micro-bentonite to the clayey sand specimen decreased the axial strain at failure. The clayey sand sample with the addition of 2% micro-bentonite at 1d of maturation reaches its maximum optimum at an axial stress of 3.3 kg/cm2 for an axial strain of 0.12%. The clayey sand sample with the addition of 3% micro-bentonite at one day of maturation reaches its maximum optimum at axial stress of 3 kg/cm2 at axial strain of 0.13%. Axial strain at failure decreases with the micro-bentonite content. The clayey sand sample with the addition of 3% micro-bentonite at 7d curing times reaches its maximum optimum at an axial stress of 3.5 kg/cm2 for axial strain of 0.13%. The clayey sand sample with the addition of 3% bentonite at 28d of maturation reaches its maximum optimum at an axial stress of 3.5 kg/cm2 for an axial strain of 0.13%. Adding 3% micro-bentonite to the sample increased the axial stress by 0.5 kg/cm2 (3 kg/cm2/3 kg/cm2) in 7 days curing times. It can be assumed that the addition of 3% micro-bentonite to clayey sand stabilizes the value of axial stress at failure regardless the curing time. However, it can also be assumed that the axial stress and strain at failure stabilize after 7d. There is not much difference between the curve representing the addition of 3% bentonite at 7d of cure and the curve representing the addition of 3% bentonite at 28 days of cure.The results suggest that the addition of micro-bentonite can be used to strengthen clayey sand and improve their ability to resist deformation and failure under heavy loads. The clayey sand curve at 1d of cure increases gradually until it reaches its maximum with an axial stress of 3 kg/cm2 for an axial strain of 0.18%. The clayey sand specimen with the addition of 1% nano-bentonite content at 1 day curing reaches its maximum optimum at axial stress of 4.5 kg/cm2 for an axial strain of 0.17%. The clayey sand sample with the addition of 2% nano-bentonite content at 1d curing reaches its maximum optimum at axial stress of 5 kg/cm2 for axial strain of 0.21%. The clayey sand sample with the addition of 3% nano-bentonite content at 1 day curing reaches its maximum optimum at axial stress of 6 kg/cm2 for axial strain of 0.25%. The higher additive content the larger axial stress at failure.The Fig. 2 for axial strain of 0.15%. The clayey sand sample with the addition of 3% nano-bentonite at 28d curing reaches its maximum optimum at axial stress of 7 kg/cm2 for axial strain of 0.15%. It can be seen again that at 7d curing, the strength of clayey sand sample with the addition of 3% nano-bentonite content stabilizes. Observations suggest that the addition of nano-bentonite improves the resistance of clayey sand acting as a binder. The results on Fig. The clayey sand sample with the addition of 3% nano-bentonite content at 7d curing reaches its maximum optimum at axial stress of 7 kg/cm2 respectively. On 7d, the secant modulus at 50% of peak stress of micro-bentonite and nano-bentonite are 2300 and 4500 kg/cm2 respectively. A reduction in the secant modulus at 50% of peak stress are observed for micro-bentonite while that of nano-bentonite doubles. On 28d, the E50 of micro-bentonite and nano- bentonite are 2300 and 6000 kg/cm2 respectively. A further decrease in the secant modulus at 50% of peak stress is observed for the micro-bentonite while that of the nano-bentonite continues to increase. A decrease in secant modulus at 50% of peak stress over time is observed for micro-bentonite. However, a strong increase in secant modulus at 50% of peak stress over time is observed for nano-bentonite. On 1d, the E50 is 2000 kg/cm2 and approaching 6000 kg/cm2 on 28 d. The E50 is thus 3 times higher on day 28 than on 1d. Observations suggest that nano-bentonite with 3% additive has higher secant modulus at 50% of peak stress over time, whereas that of micro-bentonite degrades.On 1d, the secant modulus at 50% of peak stress (E50) of micro-bentonite and nano-bentonite are 2500 and 2000 kg/cmThe results on the bar graph in Fig. 2. With the addition of 1% of micro-bentonite and nano-bentonite it reaches respectively 2500 and 3000 kg/cm2 at one day of curing time.The clayey sand without additives achieves E50 secant modulus at 50% of 1500 kg/cmThe E50 modulus of the soil treated with micro-bentonite and nano-bentonite is higher than that of clay sand specimen.Examining the morphology of clayey sand specimens with micro- and nano- bentonite, Scanning Electron Microscopy (SEM) showed the cementation mechanism that leads to improving the mechanical properties of soil samples. SEM involves using an electron beam to scan the surface of a sample and produce its two-dimensional image. Untreated and treated soils have very complex structure where it is not easy to demonstrate the presence of micro- and nano-particles using SEM although micro- and nano- bentonites were observe in various SEM images shown in Figs. Scanning electron microscope analysis of the nano-bentonite particles in Fig. In order to confirm the nano-bentonite additive reaches the maximum strength improvement in clayey sand specimens including 3% nano-bentonite, the samples were evaluated with SEM. SEM images provided good observation about the nano-bentonite process and change of surface compositions in different clayey sand soil samples.X-ray diffraction (XRD) testing is a method used to study the crystal structure of a substance. In order to confirm the nano-bentonite additive reaches the maximum improvement in clayey sand specimen including 3% nano-bentonite, samples were evaluated with XRD. The results showed that micro-bentonite is characterized by a very fine and homogeneous crystalline structure. The diffraction peaks observed on the graph of Fig. In general, the results of the X-ray diffraction (XRD) test confirm that micro-bentonite is indeed a smectite clay and show that the crystal structure of bentonite is very fine and homogeneous. This kind of crystal structure is very favorable for geotechnical applications, as it can improve the shear strength and stability of soft soils. Figure Observing the results of the X-ray diffraction test for nano-bentonite shows distinct and well-defined peaks at specific diffraction angles. The diffraction peaks on the graph in Fig. The results of this test show that nano-bentonite has a different crystal structure than conventional micro-bentonite, which may explain the differences in the properties of these two materials. Indeed, nano-bentonite is mostly composed of Quartz and has a larger surface area, which can lead to improved bonding properties when used to improve clayey sand.In order to confirm the nano-bentonite additive reaches the maximum strength improvement in clayey sand specimen including 3% nano-bentonite, XRD tests were performed. In conclusion, the results of the X-ray diffraction test shows that nano-bentonite has a well-defined crystal structure different from that of conventional micro-bentonite, which may explain the differences in their properties. The observations from this test are very important for the improvement of clayey sand, as they show that nano-bentonite can be a promising material for this application due to its unique properties.According to the literature review, most researchers used micro bentonite in soil improvement; also, they conducted the powder method for soil stabilization. This research concluded that micro-bentonite can be a viable option for soil stabilization, but it requires more research and optimization to determine the optimal conditions and processes for different soils and applications. This research also highlighted the potential benefits of using nano-bentonite in a suspension condition as a novel additive for soil improvement.This research will promote utilization of micro- and nano-bentonite for soil improvement techniques. This study confirmed the role of micro- and nano- bentonite in nano soil-improvement techniques. In conclusion, our results showed that nano-bentonite can be considered as more effective additive to improve the properties of soft soils compared to micro-bentonite, thanks to its spherical shape, its smooth and uniform surface, its ability to increase soil stability and its higher permeability. These properties could make it superior to traditional bentonite as an additive to improve soft soils. Atterberg limit tests have shown that nano-bentonite additives can reduce plasticity index and improve clayey sand consistency. Our results show that nano-bentonite is more effective over long term in improving the properties of clayey sand than micro-bentonite when combined with 3% content. According to results of this research, the use of 3% nano-bentonite is recommended for significant improvement of untreated clayey sand.The results showed that nano-bentonite suspension significantly improved the strength and stiffness of the soil-bentonite mixture by forming gel that filled the pores and bonded the soil particles. Nano-bentonite also acted as a nucleation site, enhancing the durability and strength of the mixture. Nano-bentonite suspension improved soil specimens, which filled the voids by the gels. This developed a new method for soil improvement better than the powder method that different researchers used before. Nano-bentonite as a type of nano-additives can be used in soil improvement techniques in clayey sand. This is one of few studies where micro- and nano-bentonite were directly applied in soil stabilization. This research was done in laboratory conditions. Future work is required to do in large scale and field tests.


In 2020, Wales experienced some of the highest rates of confirmed COVID-19 cases in Europe. We set up a serosurveillance scheme using residual samples from blood donations to inform the pandemic response in Wales.To identify changes in SARS-CoV-2 antibody seroprevalence in Wales by time, demography and location.Residual samples from blood donations made in Wales between 29 June 2020 and 20 November 2022 were tested for antibodies to the nucleocapsid antigen (anti-N) of SARS-CoV-2, resulting from natural infection. Donations made between 12 April 2021 and 20 November 2022 were also tested for antibodies to the spike antigen (anti-S) occurring as a result of natural infection and vaccination.Age-standardised seroprevalence of anti-N antibodies in donors remained stable (4.4–5.5%) until November 2020 before increasing to 16.7% by February 2021. Trends remained steady until November 2021 before increasing, peaking in November 2022 (80.2%). For anti-S, seroprevalence increased from 67.1% to 98.6% between May and September 2021, then remained above 99%. Anti-N seroprevalence was highest in younger donors and in donors living in urban South Wales. In contrast, seroprevalence of anti-S was highest in older donors and was similar across regions. No significant difference was observed by sex. Seroprevalence of anti-N antibodies was higher in Black, Asian and other minority ethnicities (self-reported) compared with White donors, with the converse observed for anti-S antibodies.We successfully set up long-term serological surveillance of SARS-CoV-2 using residual samples from blood donations, demonstrating variation based on age, ethnicity and location. What did you want to address in this study?Serosurveillance can provide vital information about the circulation of disease in a population as a whole, including those who may have experienced mild or asymptomatic disease. Our surveillance system aimed to monitor the SARS-CoV-2 antibody responses of the Welsh blood donor population over time, in order to identify trends in natural exposure to the virus and vaccination.What have we learnt from this study?The proportion of donors with vaccine-mediated (anti-S) antibodies increased over time with the majority having protection by the end of the study. The proportion of donors who had experienced a natural infection (indicated by anti-N) reached 80% by November 2022, suggesting high levels of disease transmission. Age, ethnicity and location of residence have a significant impact on the presence of antibodies, however sex does not.What are the implications of your findings for public health?Serosurveillance is an effective means of monitoring exposure to SARS-CoV-2 within a population. Demographic variations have a significant impact on SARS-CoV-2 antibody prevalence. Understanding differences in immunity across a region/country allows for targeted public health action.The United Kingdom (UK) experienced some of the highest cumulative coronavirus disease COVID-19) case numbers 9 case nu in EuropSurveillance of COVID-19 in Wales was carried out, primarily by following trends in: people who had a positive SARS-CoV-2 PCR (PCR) test result; people hospitalised with COVID-19; and people who died within 28 days of a confirmed PCR test. Changes in testing policy during the epidemic in Wales led to bias in case counts and rates, two of the key surveillance metrics. Furthermore, hospitalisation and mortality trends only included those with severe clinical features, excluding those with mild or asymptomatic infection.Serological surveillance for antibodies has been shown to be an effective method of monitoring the spread of infection in a population -10. SeroThroughout the pandemic, the Welsh Blood Service (WBS) has continued to collect blood donations from volunteer donors. Blood donors are a convenient sample population for serological surveillance as they are accessible and provide sampling opportunities as part of blood product retrieval. In order to provide information on community exposure to SARS-CoV-2 to inform the pandemic response in Wales, we set up a serosurveillance system using residual samples from donated blood. We describe the setting up of the serosurveillance project and present data on the seroprevalence of SARS-CoV-2 antibody in Welsh blood donors, by time, demography and location.The establishment of this surveillance system was a collaboration between WBS, Swansea Bay University Health Board (SBUHB) and Public Health Wales (PHW), beginning in June 2020, with Cwm Taf Morgannwg University Health Board (CTMUHB) joining in January 2022. A steering group, comprising of members from each organisation, met regularly throughout the project. The system was composed of three stages: (i) sampling; (ii) testing; (iii) data analysis and feedback.The population comprised volunteer donors aged 17 and over, donating whole blood between 29 June 2020 and 20 November 2022, to WBS, part of the National Health Service in Wales. Quota sampling was used with an aim of including the first one hundred samples each day. Demographic and clinical information relating to the donor and donation were recorded. The demographic information provided to PHW was not personally identifiable, however a donor key was supplied to identify repeat donations from an individual. Donor characteristics provided were age, sex, ethnicity and area of residence (Lower Layer Super Output Areas (LSOA)). Initially, sex was defined as self-reported current gender. However, in 2022, WBS updated this to sex assigned at birth. All blood donors were asked to self-report their ethnicity, with 3% responding ‘no stated ethnicity’.Samples were waste products from blood manufacturing that would normally be discarded. Room temperature samples were delivered to SBUHB or CTMUHB by courier on a daily basis with an electronic list of samples included.Antibody testing was carried out at the biochemistry laboratories at SBUHB and CTMUHB. Initially, samples were only tested for antibodies to the nucleocapsid antigen . However, donations made after 12 April 2021 were also tested for antibodies to the spike antigen . Samples negative for anti-N and anti-S antibodies usually indicate the absence of recent SARS-CoV-2 infection or vaccination; those positive for anti-N and anti-S antibodies indicate recent natural infection; and those positive for anti-S antibodies only indicate recent vaccination without infection. Testing was carried out using the Roche Immunoassay . Results were determined by employing electrochemiluminescence as the output signal with a positive cut-off index (COI) of 1.0 for the anti-N antibodies and 0.8 U/mL for anti-S antibodies. Local verifications of the antibody assays were performed on the e801 platform. Precision was assessed by performing replicate measurements on positive and negative patient pools across 5 days (n = 25). The anti-N within-run precision coefficient of variation (CV) was 1.9% at 0.10 COI and 1.7% at 13.5 COI; between-batch CV was 3.6% at 0.10 COI and 2.3% at 13.5 COI. The anti-S within-run CV was 5.6% at 0.55 U/mL and 2.8% at 44.7 U/mL; between-batch CV was 7.2% at 0.55 U/mL and 2.9% at 44.7 U/mL.Assay specificity (true negativity) was assessed by analysing anonymised samples stored from 2019 (pre-pandemic), with anti-n = 100%  and anti-S = 100% . Sensitivity (true positivity), was assessed by analysing samples collected from donors > 14 days after a positive SARS-CoV-2 PCR result or vaccination (anti-S only), with anti-n = 94%  and anti-S = 100% .Pre- and post-vaccination samples were compared from 15 SARS-CoV-2 naïve subjects. All were anti-N and anti-S negative before vaccination and only anti-S positive, 14 days after vaccination .Data files were uploaded to a secure portal to be accessed by the surveillance team at PHW. Staff from SBUHB and CTMUHB uploaded files of test results, each with a unique identifier and staff from WBS uploaded files containing donor demographic information and files containing lists that matched test results with demographic details. Staff from PHW then extracted the data from the portal and aggregated data at 4-week periods. Stata version 14   was usedIn order to provide a headline surveillance indicator, cell formulae in Microsoft Excel were used to calculate direct age-standardised seroprevalence using mid-year 2019 Welsh population estimates, by age group , which accounted for 79% of the population. This population structure was used to calculate the age-standardised value throughout. However, there were no donors in the 70–85-year-old group during period 1, likely due to national lockdown regulations.Data were analysed by time, demography and location. For ethnicity, cumulative, as opposed to rolling seroprevalence was calculated, with black, Asian and minority ethnic groups being grouped as one due to low sample size. Lower Layer Super Output Areas were used to determine residential local authority (LA) and then LAs were grouped by the areas covered by local resilience fora: South Wales, Gwent, Dyfed and North Wales. Seroprevalence was presented as a percentage and was calculated by dividing the number of samples with a positive result for that period by the total number of samples included in the analysis for that period. Stata version 14 was used to calculate 95% binomial confidence intervals. Time trends of changes in seroprevalence by age group, sex and area of residence were plotted.A short surveillance report was produced for each 4-week period, which was disseminated to key stakeholders involved in the pandemic response in Wales, such as the Health Minister for Wales.In samples that were tested for both anti-N and anti-S antibodies, the presence of both were quantified. Repeat donors were identified in order to investigate changes in positivity over time.A total of 71,827 samples from 45,649 donors , male: 43.3% , median age: 46 years (33–57)) were tested and included in the analysis. A small proportion of samples  were excluded due to the inability to link to demographic information, missing results or donors giving convalescent plasma. The latter were excluded as they were invited to donate as a result of a recent SARS-CoV-2 infection. Thirty-one 4-week periods were included with an average of 2,317 samples per period . Periods 1–5 saw a relatively stable prevalence of anti-N antibodies . A rapid increase was then seen between period 6 , period 7  and period 8  before levelling out for the next 10 periods . From this point, a steep upward trend was seen, peaking in the final period (period 31) . For anti-S antibodies, the direct age-standardised rate showed a marked increase from 67.1% (95% CI: 61.7–72.7) in period 11 to 98.6% (95% CI: 94.5–100) in period 16. A steady increase followed, reaching 99.1% (95% CI: 94.3–100) by period 22 and the prevalence continued to be above 99% for the remainder of the study .Males and females had an equal cumulative seroprevalence of anti-N antibodies , over the course of surveillance. This was also seen for anti-S antibodies .Significant differences in antibody prevalence  do not overlap) were found between age groups. For anti-N antibodies, seropositivity remained highest in those aged 17–29 years throughout the study  compared to other groups aged 40–49 years ; 30–39 years ; 50–59 years ; and 60–85 years . The highest prevalence of anti-S antibodies was seen in 60–85-year-olds  compared to age groups 50–59 years , 40–49 years , 30–39 years  and lastly 17–29 years . For both anti-N and anti-S antibodies, all age groups appeared to follow a similar pattern over time .Data were aggregated over 4-week periods. Numbers in brackets refer to the corresponding 4-week period. Ethnicity has been recorded for approximately 93% of all blood donors since the beginning of 2020, with 2% reporting to be of Black, Asian or other minority ethnicity. Seroprevalence of anti-N antibodies was significantly higher in people self-reporting as Black, Asian or other minority ethnic group  than those self-reporting as White . Seroprevalence of anti-S antibodies was slightly higher in people of White ethnicity  than those of Black, Asian and other minority ethnicities .Seropositivity of antibodies varied significantly depending on region of residence. Persons living in South Wales and Gwent had the highest cumulative rates of anti-N antibodies at 29.1% (95% CI: 28.6–29.6) and 28.0% (95% CI: 27.2–28.8), respectively. Anti-N antibodies rates were significantly lower in those living in North Wales .For anti-S antibodies, the highest cumulative positivity was seen in Gwent , followed by South Wales , North Wales  and Dyfed Powys . Between periods 11 and 31, 44,298 samples were tested for both anti-N and anti-S antibodies . A slighDue to repeat donations, 17,180 donors were tested between two and eight times for anti-N antibodies over the 31 periods. In addition, 9,003 donors were tested between two and six times for anti-S antibodies over the 21 periods. The results for repeat donor testing can be seen in the This serosurveillance system has successfully provided information about variation in seroprevalence of SARS-CoV-2 antibody in blood donors residing in Wales, by time, demography and location. Investigating both anti-N and anti-S antibodies to determine the cause of seroconversion corroborates data gathered through the monitoring of vaccine coverage and provides useful information on the distribution of susceptible individuals. Using a waste product from blood manufacturing is a low-cost method of acquiring blood samples.Changes in antibody prevalence over time, illustrated by this study, are consistent with known variation in disease transmission and vaccination uptake in Wales. The initiation of the steep increase of anti-N antibodies corresponds with the emergence of the SARS-CoV-2 Omicron variant  lineage designation (B.1.1.529)) (27 November 2021 ), which Our key findings are generally in agreement with other reports of SARS-CoV-2 antibody within both blood donors and other populations. Young adults have frequently been found to have a higher rate of antibodies resulting from natural infection than their older counterparts, in many countries in Europe . However, the majority of the population of Wales also live in South Wales (43% based on mid-2020 population data ). This sInterpretations of the repeat donor data are limited due to unknown infection and vaccination histories. It is possible that individuals could be re-infected between donations, thus appearing to have continued to hold antibodies when they have in fact seroreverted and seroconverted. Therefore, the true occurrence of seroreversion may not be being represented. This could be overcome by data linkage to other health databases.This surveillance system was successful in detecting changes in seroprevalence of antibodies to the nucleocapsid and spike antigens of SARS-CoV-2 over time. This information compliments other surveillance data on cases and vaccination uptake to provide an ongoing picture of the COVID-19 epidemic in Wales, informing its prevention and control through targeted testing and vaccination initiatives.


Few studies have been conducted to analyze the prognostic factors of AE. The study aimed to explore the values of diagnosis and treatment evaluation by 18F-FDG PET and preliminarily discussed the potential value in predicting the prognosis of AE patients.mean) and maximum standardized uptake value (SUVmax) of brain relative regional metabolism (BRRM) were quantified in the case and control groups according to the anatomical automatic labeling (AAL) partition. The main statistical method was the Kruskal–Wallis test. Finally, the simple linear regression method was used to analyse the relationships between 18F-FDG PET parameters and the modified Rankin Scale (mRS) scores before and after treatment.AE patients underwent 18F-FDG PET/CT and magnetic resonance imaging (MRI). There were two steps to analyse 18F-FDG PET imaging data. The first step was visual assessment. The second step was to analyse 18F-FDG PET parameters using Scenium software (Siemens Molecular Imaging Ltd). The mean standardized uptake value (SUVp = 0.001). The number of lesions on PET was positively correlated with the mRS scores before and after treatment, and the correlation before treatment was more significant. Before treatment, the SUVmean of the left occipital lobe was the most remarkable  factor associated with the mRS score, and the correlation was negative. With regard to prognosis, the SUVmax of the MTL was the most notable  factor associated with the mRS score after treatment, and the correlation was positive.The results on 18F-FDG PET showed that visual assessment abnormalities were in the mesial temporal lobe (MTL) (70.8%), , basal ganglia (62.5%), frontal lobes (37.5%), occipital lobes (29.2%), and parietal lobes (12.5%). The positive rate of abnormalities on 18F-FDG PET was more sensitive than that on MRI (95.5% vs 32.2%, max of MTL hypermetabolism may serve as a prognostic biomarker in AE.18F-FDG PET could be more sensitive and informative than MRI in the early phases of AE. The common pattern of AE was high MTL metabolism on 18F-FDG PET, which was associated with hypometabolism of the occipital lobe, and the number of lesions on PET before treatment may be significant factors in assessing disease severity. The SUVThe online version contains supplementary material available at 10.1186/s12880-023-01148-6. Autoimmune encephalitis (AE) is a non-infectious, immune-mediated inflammatory disease of the cerebrum parenchyma; this subacute presentation is highlighted in the Graus criteria and is a hallmark of the disorder, which is different from acute encephalitis developing as a rapidly progressive encephalopathy  –5. RecenImaging examinations of AE are based on magnetic resonance imaging (MRI), which can rule out stroke, tumours, and other infectious encephalitides. 2-deoxy-2-[18F] fluoro-D-glucose positron emission tomography (18F-FDG PET) is used as an auxiliary tool to be performed with more sensitivity and information related to brain abnormalities  when theA total of 32 patients with AE were retrospectively reviewed from the Second and First Affiliated Hospitals of Harbin Medical University between January 2017 and June 2022. All patients fulfilled the clinical diagnostic criteria and were positive for AE-related antibodies in the serum or cerebrospinal fluid (CSF). Thirty-one patients underwent MRI, and 24/31 patients underwent 18F-FDG PET (both anti-GAD65 and anti-LGI1 patients were re-examined after treatment in one year). Neuroimaging examinations  were carried out in the acute and subacute stages after symptom onset. For the group analysis of 18F-FDG PET imaging, we identified 101 healthy controls without neurological anomalies, dividing them into two groups  findings for individual patients and the comparison of the results are presented in Table .Table 1All 32 patients underwent serum and CSF antibody testing, including tests for classic paraneoplastic antibodies  and N-methyl-D-aspartate receptor (NMDAR), leucine-rich glioma inactivated-1 (LGI1), contacting-associated protein-2 (CASPR2), gamma-aminobutyric acid receptor (GABABR), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), and glutamic acid decarboxylase 65 (GAD65) antibodies. Serum and CSF samples were analyzed using cell-based assays , immunohistochemical analyses in the neuroimmunology laboratory of the Peking Union Medical College Hospital and Heilongjiang Kingmed for Clinical Laboratory.The MRI scanner was a 3.0 Tesla Discovery 750w MRI . The standard MRI protocols included T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), fluid-attenuated inversion recovery (FLAIR), and diffusion-weighted imaging (DWI). For T1WI , T2WI , FLAIR , and DWI , axial images were obtained, and the slice thickness was 5 mm. Two experienced radiologists independently evaluated the MRI results. If there was obvious discordance at the beginning of the evaluations, an informed consensus was achieved.The PET/CT scanner was a Siemens Biograph 64 time-of-flight scanner. All patients were asked to fast for at least 6 h, and fasting blood glucose levels could not exceed 8 mmol/L. The injection dose was 0.12 mCi/kg, and the imaging agent was 18F-FDG. After injection, they were required to rest quietly and were isolated in a dedicated room to ensure minimal auditory and visual stimulation. The brain and whole-body imaging acquisition time was 40 min after injection. The brain acquisition time was 3 min/bed, and the speed of the whole body acquisition was 1.5 mm/s. Slice thickness was 3 mm and 1 mm.Visual assessment was performed by two board-certified radiologists (10 years). 18F-FDG PET image analysis should be performed by drawing the region of interest (ROI) and then calculating the SUV and asymmetric index (AI) :\documenmean), standard deviation of the SUVmean (SUVmeanstd), maximum standardized uptake value (SUVmax), and standard deviation of the SUVmax (SUVmaxstd) of each brain ROI . The mA score of 1 was given for a focal anomaly in a lobe or increased uptake in the basal ganglia, and a score of 0 was given for the absence of a lobar anomaly or increased uptake in the basal ganglia through Scenium analysis.The modified Rankin Scale (mRS) scores were used to assess neurological disability at the onset and the last follow-up for this disorder. The mRS scores ≤ 2 indicated a good outcome, and the mRS scores of 3 to 6 indicated a poor outcome. The relationships among 18F-FDG PET parameters, severity degrees of the disease, and the outcome at the last follow-up after treatment were assessed.t test or nonparametric Mann‒Whitney U test. The Kruskal‒Wallis test was used to analyze multiple groups of constant variable comparisons. The relationships between continuous variables of SUVS  and the mRS scores (before and after treatment) were explored by simple linear regression. A two-tailed p value less than 0.05 (p < 0.05) was considered statistically significant.SPSS 25.0 software package for Windows (IBM Corp) and GraphPad Prism 9.4.1  were used for statistical analysis and charts. Categorical variables were compared and analyzed by Fisher’s exact test. Data are presented as the mean ± SD for continuous variables with a normal distribution, and non-normally distributed variables are expressed as the median (interquartile range [IQR]). Continuous variables were compared using the All patients signed informed consent forms, and the study was approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University (number KY2022-188).p = 0.005) (Table n = 2) (p = 0.001). EEG, blood, and CSF analyses were performed before treatment, and all CSF bacterial and viral cultures were negative. There were statistically significant differences in EEG and CSF-TPC among the groups , because normal results accounted for a portion. Evidence of inflammation was verified in routine blood test results, including WBC↑ , NUET%↑ , LYMPH%↓ , and CPR↑ . Tumours were identified in 7 patients, including lung carcinoma in 6 anti-HU patients and one ovarian tumour in an anti-HU and Ri patient. The MRI was completed at a median of 8.5 days , and 18F-FDG PET was completed at a median of 30 days . There was a significant difference in the duration of symptoms to imaging between MRI and 18F-FDG PET/CT (p = 0.001).The average age of the anti-NMDAR group was close to 30 years old, the others were close to 60 years old (5) Table . Seizurep = 0.047). The prognosis was obviously improved, and the mRS score of 1  was dramatically decreased after treatment .The proportion of patients with an mRS score of 4 37.5%, 12/32) was the highest before treatment. All patients accepted the first line of treatment, and long-term immune treatment was performed in four patients  (/32 was tP = 0.001)   Fig. . Followi01) Fig. A. ParietThe MRI and 18F-FDG PET/CT  manifestations in the case groups are summarized in Table . The 18Fmax, the results revealed significantly high uptakes of the left inferior frontal gyrus , left inferior and middle temporal gyri in the anti-GABABR group compared with the certain group , and the results of the former group were higher. There was a significant difference (p = 0.023) in the right fusiform gyrus between the anti-GABABR and anti-NMDAR groups  between the anti-GABABR group and the certain group, accompanied by higher SUVs in the anti-GABABR group. The hypometabolism of the right middle occipital gyrus was significantly different between the anti-NMDAR and anti-LGI1 groups (p = 0.018), with that being lower in the former group. There were significant differences in the left supramarginal gyrus and right parietal lobe in the anti-GABABR group, the anti-HU group (p = 0.016), and the certain group (p = 0.030); the former was lower.For the SUVmean and SUVmax of the parietal and occipital lobes in the anti-NMDAR group were lower than those in control group-1, without hypermetabolism of the frontal lobe. The difference was the hypermetabolism of the unilateral hippocampus and cingulate gyrus in patient (No. 2). The other groups were also compared with control group-2, and the top four affected sites were the MTL (hippocampus), basal ganglia, other parts of the temporal lobe, and frontal lobe.The SUVP > 0.05). The correlations were positive, which was more significant before the treatment  and occipital lobes  were negatively correlated with the mRS scores before treatment , amygdala, and frontal gyrus  were positively correlated with the mRS scores before treatment, and the SUVmean of the left occipital lobe was the most remarkable result.As confirmed, the increased mRS scores before and after treatment might be associated with the number of lesions on 18F-FDG PET before treatment P > 0.0. The cormean and SUVmax of the MTL, frontal lobe, basal ganglia and parietal lobes before treatment on 18F-FDG PET were positively correlated with the mRS scores after treatment  factor.With respect to prognosis. The SUVent Fig. , and themean and SUVmax of the parietal and occipital lobes were lower than controls in the anti-NMDAR group, which were in accordance with the results of Liu X et al. [There were three major highlights and clinical implications in our study. First, it was revealed that the most vulnerable site was the MTL  in AE patients, which showed hypermetabolism by semi-quantitative brain 18F-FDG PET. The results were more convincing because of the large number of controls. The basal ganglia was the second most involved area, which was typical in anti-GABABR, LGI1, HU, and PNMA2 + /Ma2/Ta types in our study. The results of other lobes metabolism were as follows: the metabolism of the frontal lobe in the anti-GABABR group was higher, and the SUVX et al. –22. SecoX et al. , whereasX et al. . 18F-FDGmax of the MTL was the most notable factor associated with the mRS scores after treatment.Third and most importantly, 18F-FDG PET parameters were used to evaluate the severity degree and prognosis. The numbers of focuses on 18F-FDG PET before treatment were more important factors in association with the mRS scores before and after treatment, which was rarely reported in previous literature. It is easy to explain that the more parts of the cerebral cortex involved, the worse the ability to recover function, as seen in our anti-NMDAR and anti-PNMA2 + /Ma2/Ta patients who developed encephalitis. Hypermetabolism of the MTL was common in imaging diagnosis, accompanied by hypometabolism of occipital or parietal lobes. This feature aggravates the severity of this disorder. We also found that the SUVmean of the MTL in the anti-GABABR group was higher than that in the other groups, which might be a reminder that the former more easily involves the MTL. Meanwhile, this manifestation might combine to trigger different types of tumours in the anti-HU group, which was confirmed in this study. Metabolic changes on 18F-FDG PET in the extralimbic regions, consisting of the basal ganglia and occipital, parietal, and frontal lobes. Our three patients in the anti-LGI1 group, who also had faciobrachial dystonic seizures (FBDs); two patients in the anti-GAD65 group; and four patients in the anti-HU group without focal motor status epilepticus (FMSE) all showed hypermetabolism of the basal ganglia, which was as described in the previous literature [The hypermetabolism of the MTL was the most remarkable feature in our study diagnosed with anti-GABABR, LGI1, HU, anti-Ma and anti-Ta, and anti-NMDAR encephalitis, which was similar to prior reports , 25–30. terature –36. The terature  studied It is worth noting that multiple focal infiltrates of inflammatory cells lead to the development of neuronal hyperexcitability and that myoclonic jerks may arise from an atypical propagation of neuronal activity along various networks. Such propagation may differ from that observed in typical motor seizures, resembling the FBDS . Our antmax of the MTL was the most notable result in six types of AE for prognosis, which was different from one type of antibody. Future prospective studies will be required to verify these findings and explore pathogenic mechanisms.Generally, previous studies , 42 haveThis study is limited by its retrospective nature and selection bias. Twenty-two patients only underwent 18F-FDG PET in the acute and subacute phases of disease, and two patients with anti-LGI1 and anti-GAD65 group underwent 18F-FDG PET after 1 year of treatment; thus, it will be difficult to evaluate treatment effects. Further prospective and longitudinal cohort studies should be performed.mean of the occipital lobe, and the number of lesions on PET before treatment may be significant factors in assessing disease severity. The increasing SUVmax of the MTL may serve as a prognostic biomarker in AE. Future prospective studies are required to verify these manifestations and to identify more accurate prognostic factors.In summary, this study provided detailed descriptions of distinct cerebrum metabolic patterns related to acute and subacute phases of AE on 18F-FDG PET, which was more sensitive than MRI. The common pattern of AE was high MTL metabolism on 18F-FDG PET, which was associated with a decreasing SUVAdditional file 1: Supplemental Fig. 1 Other results of comparative BRRM across different sites in the case groups. Supplemental Fig. 2 Simple linear regression, to evaluate the relationship among SUVmean and SUVmax of BRRM and mRS scores before treatment, superior temporal lobe(R), caudate nucleus(R), middle frontal gyrus, orbital part (R), pallidums and basal ganglia had the positive relationship before treatment. Supplemental Fig. 3 Simple linear regression, to evaluate the relationship among SUVmean and SUVmax of BRRM and mRS scores after treatment. SUVmean and SUVmax of MTL had the positive correlation with the mRS score after treatment.Additional file 2: Supplementary Table 1. The SUVmax of case groups according to AAL standards. Supplementary Table 2. The SUVmean of case groups according to AAL standards. Supplementary Table 3. The SUVmaxstd of case groups according to AAL standards. Supplementary Table 4. The SUVmeanstd of case groups according to AAL standards. Supplementary Table 5. The SUVmean of normal 19-44 years old group according to AAL standards. Supplementary Table 6. The SUVmax of normal 19-44 years old group according to AAL standards. Supplementary Table 7. The SUVmeanstd of normal 19-44 years old group according to AAL standard. Supplementary Table 8. The SUVmaxstd of normal 19-44 years old group according to AAL standard. Supplementary Table 9. The SUVmean of normal 45-70 years old group according to AAL standard. Supplementary Table 10. The SUVmax of normal 45-70 years old group according to AAL standard. Supplementary Table 11. The SUVmeanstd of normal 45-70 years old group according to AAL standard. Supplementary Table 12. The SUVmaxstd of normal 45-70 years old group according to AAL standard.


An initial examination was performed preoperatively, and at 6 weeks and 4, 6, and 12 months. The primary endpoint was the subjective International Knee Documentation Committee (IKDC) score to measure participants’ subjective perceptions. Secondary endpoints included objective knee function assessed by IKDC, instrumented knee laxity measurements, isokinetic strength tests of the knee extensors and flexors, Lysholm Knee Score, Tegner Activity Score, Anterior Cruciate Ligament—Return to Sport after Injury Score, and quality of life determined by Short Form-36 (SF36). Results: There were no statistically significant or clinically meaningful differences in IKDC scores between the two study groups  −1.39 to 7.97, p = 0.03 for evidence of non-inferiority of brace-free compared with brace-based rehabilitation). The difference in Lysholm score was 3.20 (95% CI −2.47 to 8.87); the difference in SF36 physical component score 0.09 (95% CI −1.93 to 3.03). In addition, isokinetic testing did not reveal any clinically relevant differences between the groups (n.s.). Conclusions: Brace-free rehabilitation is non-inferior to a brace-based protocol regarding physical recovery 1 year after isolated ACLR using HT autograft. Consequently, the use of a knee brace might be avoided after such a procedure. Level of Evidence: Level I, therapeutic study.Purpose: The postoperative use of a rehabilitative knee brace after isolated primary anterior cruciate ligament (ACL) reconstruction (ACLR) using a hamstring tendon (HT) autograft is controversial. A knee brace may provide subjective safety but can cause damage if applied incorrectly. The aim of this study is to evaluate the effect of a knee brace on clinical outcomes following isolated ACLR using HT autograft. Methods: In this prospective randomised trial, 114 adults  underwent isolated ACLR using HT autograft after primary ACL rupture. Patients were randomly assigned to wear either a knee brace ( Anterior cruciate ligament (ACL) ruptures are among the most common musculoskeletal injuries in athletes, adolescents, and the active population. Arthroscopically assisted ACL reconstruction (ACLR) is one of the top 10 orthopaedic surgical procedures performed annually worldwide . Knee brThere is conflicting evidence on the effectiveness of postoperative bracing after isolated ACLR ,24,25,26Therefore, a randomised trial comparing brace-free and brace-based rehabilitation approaches was designed. The primary outcome measure was the difference in subjective IKDC scores (sIKDC) 12 months after isolated ACLR using HT autograft. The secondary objective was to investigate the isokinetic strength across different muscle groups as a possible measure for monitoring the effectiveness of rehabilitation. It was hypothesised that brace-free rehabilitation is not inferior to brace-based rehabilitation in terms of functional and health-related quality of life outcomes.This single-centre randomised controlled non-inferiority trial with parallel-group assignment in a 1:1 ratio was carried out at a tertiary care referral trauma centre in Germany. It was conducted in accordance with the Consolidated Standards of Reporting Trials (CONSORT) and principles of the International Conference on Harmonization Good Clinical Practice (ICH-GCP). The Institutional Review Board (IRB) of the University Witten/Herdecke, Germany, approved the protocol (Reference-No. 14/2015) on 7 July 2015. The trial commenced on 1 June 2016 and was registered at the German Clinical Trial Registry (DRKS00011774).2 scheduled for isolated ACLR of a primary ACL tear using the ipsilateral HT were enrolled. Patients demanding partial meniscal resections and those with smaller meniscal lesions in the white–white zone not requiring resection remained eligible to participate, as were subjects with previous injury to the contralateral knee but normal knee function. Subjects with ligament injuries other than the ACL, previous knee ligament surgeries, severe knee osteoarthritis of Kellgren–Lawrence grade ≥ 3, cartilage replacement procedures, and necessary meniscal repair were excluded.Prior to enrolment, patients were informed about the trial purpose and procedures, and provided written informed consent. Patients aged 18–60 years with a body mass index (BMI) < 35 kg/mAll surgical procedures were performed by two advanced orthopaedic surgeons, using ipsilateral HT autograft. The patients were in a supine position, and an electric leg holder was used. Following diagnostic arthroscopy, the semitendinosus tendon was harvested via an oblique tibial skin incision, and a quadrupled single-bundle graft was then created. Only in cases of a small graft diameter (<7.5 mm), the gracilis tendon was harvested additionally to obtain a six-fold graft. First the femoral tunnel was drilled through the anteromedial portal, respecting the femoral attachment of the torn ACL. The tibial tunnel was drilled in an outside-in technique using a target device and orientating on the tibial origin of the torn ACL. Via a transtibial shuttle device the graft was pulled into the joint. An extracortical button device was used for femoral fixation . Tibial fixation was performed using a hybrid fixation technique with a bioabsorbable screw  and washer . After wound closure the position of the fixation devices and the drill holes was controlled by X-ray.An independent research assistant prepared opaque envelopes based on a computer-generated randomisation list. After surgery, the surgeon opened the envelope in operating theatre and assigned patients to the brace-based or brace-free group according to random allocation. Blinding was not possible for either the surgeon or patient because of the possibility of wearing a brace.Those in the brace-based group wore a technically identical rehabilitative four-point hard-frame knee brace  and mental component summary (MCS) scores of the Short Form 36 (SF-36) questionnaire . The SF-Subjective confidence in RTS activities was evaluated using the Anterior Cruciate Ligament—Return to Sport after Injury (ACL-RSI) score. The ACL-RSI score, with values ranging from 1 to 10, includes 5 questions on emotional well-being, 5 questions on confidence in physical performance, and 2 questions on risk assessment. Higher scores indicate a more positive psychological response. The total score was determined by adding the values of the 12 responses and then calculating their relationship to 100 to obtain a percentage.Secondary objective outcomes included the objective IKDC score (oIKDC) . The oIKBaseline data were measured at outpatient clinic before, 6 weeks, 4, 6, and 12 months after the index procedure . All meaIsokinetic strength testing was conducted on a Biodex System 3 device . Isokinetic testing proved good to high test–retest reliability and has extensively been employed as a safe method to determine muscle strength in patients after ACLR ,40,41,42Data were described according to their quality and expression as numbers, frequencies (%), means, or medians, with suitable measures of distribution , and standard deviations (SDs)). Whenever possible, 95% confidence intervals (CIs) were calculated and reported for individual estimates, differences, and ratios. Primary endpoint was the sIKDC 12 months postoperatively. We assumed non-inferiority of brace-free rehabilitation compared to brace-based rehabilitation after isolated ACLR operationalized by the difference in sIKDC scores after 12 months of follow-up. According to the American Orthopaedic Society for Sports Medicine (AOSSM) Outcomes Task Force, the reported responsiveness of the sIKDC ranges from 6.7 to 20.5 in the Minimal Detectable Change (MCD) and 3.2 to 16.7 in the Minimal Clinically Important Difference (MCID) . We simuOf 670 patients screened, 531 did not meet inclusion criteria. A total of 139 patients were recruited and randomised (71 in the intervention group and 68 in the control group) from June 2016 to April 2019 . One patBaseline demographic and clinical characteristics of patients are shown in Complications occurring post-randomisation are listed in p for non-inferiority < 0.0001). p values for non-inferiority remained at 0.0254 and 0.0001, respectively. Adhering to a threshold of 0.05, apart from discussions of lowering the level of significance to 0.005  and assuming minimal non-inferiority margins, it is statistically unlikely that our observations are compatible with chance.Brace-free rehabilitation proved to be non-inferior to brace-based rehabilitation based on an a priori-defined MCID of 9.0 .The ACL-RSI improved from 70.2 ± 18.1 points in the brace-free and 65.9 ± 19.8 points in the brace-based group after 6 months up to 70.4 ± 23.3 points in the brace-free group and 72.5 ± 20.4 points in the brace-based group after 12 months. A cut-off value of 56 points is reported  to reflep < 0.0001). With the lowest MDC and MCID of 8.9 and 10.1, as provided by the AOSSM Outcomes Task Force, p values for non-inferiority remained at 0.0044 and 0.0012, respectively.The mean Lysholm score at 12 months was 88 ± 4.0 and 85 ± 3.9 for the brace-based and brace-free groups, respectively. Hence, brace-free rehabilitation was non-inferior to brace-based rehabilitation, given a priori-defined MCID of 9.0 . Furthermore, brace-free rehabilitation was also found to be non-inferior to brace-based rehabilitation when judged based on the health-related quality of life measurements  and mean MCS scores .The mean Tegner activity scale value increased from 2.7 ± 0.6 (brace-free group) and 3.5 ± 0.6 (brace-based group) prior to injury to 5.3 ± 0.6 and 5.5 ± 0.5 at 12 months after surgery, respectively. Brace-free rehabilitation proved to be non-inferior to brace-based rehabilitation  revealed no significant difference between the groups for knee extension and knee flexion .Force production of the knee extensors and knee flexors was measured at an angular velocity of 60°/s. Maximal torque for the knee extensors increased equally in both groups from 4 to 12 months after surgery. The same trend was observed for the maximal knee flexion torque at these time-points . The mixTo differentiate strength differences between the brace-free and brace-based rehabilitation groups, the mean group differences for all isokinetic parameters were calculated with reference to the ACLR knee.The most important finding of this study was, that subjective and objective outcomes following isolated ACLR did not differ significantly between subjects wearing a brace and those not wearing a brace postoperatively. Therefore, the hypothesis of non-inferiority of brace-free versus brace-based rehabilitation was proven. Moreover, instrumented laxity measurements and isokinetic knee strength in the brace-free group proved to be non-inferior to the brace-based group. Finally, both groups rated their ACL injury related self-confidence high enough to participate in pre-injury sporting activities.Although many consider bracing an integral part of rehabilitation after ACL injuries ,48,49, LRehabilitation after HT-ACLR differs from that after BTB autografts as there is no interface between bone and tendon, resulting in a prolonged remodelling process following ACLR . TherefoIn addition, the present trial provides strong evidence that bracing after isolated ACLR using HT autograft does not lead to superior functional outcomes compared to brace-free rehabilitation.Patients in either group reported comparable results (70.4 ± 23.3 point in the brace-free group and 72.5 ± 20.4 in the brace-based group) in the ACL-RSI, rating readiness to RTS after injury .As recently highlighted, studies that minimise bias are needed to further evaluate the role of bracing in ACL injuries ,54. In tThis study is not without limitations. A significant number of patients required combined ACLR and meniscal suturing. As we excluded these patients, it remains unclear whether our results are applicable to rehabilitation after any type of combined ACLR surgery, thereby hampering external validity. Additionally, we herein report results 12 months after surgery and cannot make predictions whether our findings are sustainable. As however the early postoperative period was assessed, a longer follow-up would have been outside the aim of the current investigation. In our collective, a male predominance was seen. This is in line with other studies on ACLR, as the incidence of ACL injury is higher in men in the general population.According to the results of this study, the use of any external stabilizers after isolated ACLR using HT autograft should be avoided in clinical practice. The results reduce treatment costs and simplify the rehabilitation protocol, which partly varied up to now.Brace-free rehabilitation after isolated ACLR using HT autograft is non-inferior to brace-based protocols regarding physical recovery one year postoperatively. Patients without bracing after isolated ACLR showed no significant difference in the ACL-RSI compared to the control group in the current study. Thus, it can be concluded that patients regained equal confidence in the operated knee after 6 and 12 months, regardless of the use of a brace. It remains questionable whether bracing can also be avoided after revision and combined ACLR.


Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution. Therefore, this technology has become a powerful tool for time-lapse observations over long periods of time, enabling the elucidation of single-cell behaviors of multicellular organism morphogenesis [Light-sheet microscopy is fluorescence microscopy in which the detection and illumination optical axes are placed orthogonally. It achieves high-resolution biological imaging with a thin sheet of light from the side on the focal plane of the detection objective lens . This teogenesis ,3.The illumination and the detection machinery determine the optical properties of the light-sheet microscopy, such as spatial resolution and field of view (FOV). There is a trade-off between FOV and axial resolution due to the orthogonal geometry of the illumination and detection pathways of light-sheet microscopy. Since the excitation light is incident from the side, thus, the length of the focused beam is directly related to the FOV, and the width of the beam is directly related to the axial resolution. This means that lengthening the beam to improve FOV results in a thicker beam and lower resolution, whereas narrowing the beam to improve resolution results in a shorter beam and narrower FOV. This makes high-resolution imaging of large specimens challenging.The use of non-diffracting beams, such as Bessel ,5,6,7, AThe design of the light-sheet microscope should match the thickness of the illumination sheet and the depth of focus (DOF) of the detection objective (DO). High numerical aperture (NA) objectives can efficiently collect fluorescence signals, but they have a tight DOF. This mismatch between the light-sheet and the DOF of the DO can cause not only a loss of in-focus fluorescence signals, but also the detection of out-of-focus signals as backgrounds, reducing the signal-to-noise ratio (SNR). Moreover, when the illumination light propagates through biological samples, due to the inhomogeneous distribution of objects, bending and spreading of the light-sheet by scattering and absorption through samples leads to the lowering of signals and degrading of image contrast, decreasing the penetration deep inside tissues. For two-photon imaging over large samples, this light-sheet modulation can become a severe issue because of its quadratic dependence on two-photon excitation. Therefore, achieving high-resolution in vivo imaging over the entire sample is challenging.An extension of DOF can address this issue of illumination and detection mismatch. In this study, we used this technique to improve the wide-field two-photon microscopy for higher resolution and SNR imaging while keeping a large FOV. For this purpose, we used a low magnification 16×) and high NA (0.8) DO that provides a DOF of ~2 μm. This is narrower than the light-sheet thickness of our system, while we previously used a low magnification (10×) and middle NA (0.5) DO that gives a DOF of ~5.4 μm, covering the full light-sheet thickness. To solve the mismatch, we used a stair-step device composed of five-layer annular zones. The device has been reported to extend DOF by several times more than the conventional system [× and higz) direction  was used. A theoretical criterion of the DOF for this DO was calculated as d = λ/n(1 – cos α) =1.96 μm, where λ (525 nm) is the wavelength, n (1.33) is the refractive index, and α is the angle determined by the objective NA (0.8) [We constructed a two-photon light-sheet microscope with an extended DOF device that stretches the point spread function (PSF) of the detection system along the axial (irection A. The syNA (0.8) . The beaz-axis) of the systems with and without the device are 3.55 μm and 1.73 μm, respectively, and it indicates that the device extends the DOF two-fold  and transverse (y) direction at the beam waist  A. The peam waist B,C. The am waist B. This cam waist C. Since am waist . We firsd FWHM2D . Using td FWHM2D . The bead FWHM2D E. This id FWHM2D B. In addd FWHM2D . In orded FWHM2D A,C,E. Wed FWHM2D B,D,F. Thz-stack images with 2-μm step size, 1024 × 1024 pixels (0.8 μm × 0.8 μm/pixel), and 100-ms exposure time. The z-stack images consist of optical sections spanning > 600 μm in depth. The body trunk of the lymphatic vessel  embryos of the medaka FLT4-EGFP strain, which expresses EGFP in the lymphatic endothelial cells and some venous vascular endothelial cells . EmbryosThis study investigated the utility of the five-layered stair-step device as an extension of the DOF to achieve a wide FOV and high-resolution imaging in two-photon excitation Bessel beam light-sheet microscopy. The results showed that the lateral resolution reduction is kept small, and image quality degradation at the distal side of the beam injection can be compensated, demonstrating that the microscope with the extended DOF device allows imaging of living fish (medaka) embryos with high resolution over the entire body. The extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging of large-size multicellular organism specimens in sub-cellular resolution.Light-sheet microscopy employs separate illumination and detection pathways in which the pathways are placed orthogonally to each other. Due to this optical geometry, the spatial resolution is determined by a combination of illumination and detection components, which should thus be carefully designed. The illumination part of our microscope system is based on the two-photon excitation Bessel beam, which achieves 600–1000-μm FOV with 3–5-μm thickness when using a NA 0.3 10× magnification illumination objective lens . A BesseAn application of the stair-step device in light-sheet microscopy has been reported . They apThere are other methods for extending the DOF, such as the use of wavefront coding techniques ,22 and aAs discussed above, the illumination Bessel beam offers highly homogeneous illumination over the FOV of the microscope system while keeping its thickness. Combined with a high NA objective, it further provides high-resolution, high-contrast imaging for large samples. When the illumination light propagates through the biological samples, bending and spreading of the light-sheet by scattering and absorption through samples leads to the lowering of signals and degrading of image contrast, decreasing the penetration deep inside tissues. For two-photon imaging over large samples, this light-sheet modulation is a severe issue because of its quadratic dependence on two-photon excitation. It is expected that the original system (without the device) is sensitive to these light-sheet modulations. Actually, our results showed the image quality degradation for living samples at the distal site from the beam injection. We demonstrated image quality recovery at the distal site in the extended DOF system, although it provides similar image quality to that of the conventional setup (without the device) at the center and proximal areas of the images. Therefore, an extension of the DOF is useful for compensation of the illumination beam modulation. Therefore, two-photon light-sheet microscopy with extended depth of focus provides an easy setup for high-quality imaging of living large-sized samples.Two-photon excitation microscopy is becoming a promising tool for observing living thick and opaque tissues, and has been extensively used as a histopathological diagnostic tool for assessment of various diseases . We havexy-plane by scanning the beam along the y-direction using a Galvano mirror scanner at a rate of 100 Hz along the y-axis. This laser-induced light-sheet was detected through the detection objective lens, a tube lens with a 200-mm focal length , and an image-splitting optics system, W-View Gemini-2C . The extended DOF device  was inserted in the second pupil plane of the optical system. An sCMOS camera with a 6.45-μm pixel size with 2048 × 2048 pixels  was used for image acquisition. The images were recorded as unsigned 16-bit gray-level images. During the emission pathway, a short-pass filter at 700 nm  was inserted to cut the scattered excitation lights, and band-pass filters at 520/35 nm (center wavelength/bandwidth) (Semrock) were used. The sample chamber was made from acrylic plates and cover glasses of 0.12–0.17 mm thicknesses . The sample-mounting holder was placed on the motorized stages , controlling the translation in the xyz-directions and the angle rotation in θ along the y-axis. The z-stack image sequence was captured by moving the motorized stages along the z-direction. A program to control the camera, motorized stages, motorized filter wheel, and Galvano mirror scanner was written using Labview2015 software .The base optical system has been described previously . For illx) and transverse (y) axis were calculated. For evaluation of the microscopy resolution, yellow–green fluorescence beads  of a 200-nm diameter that were embedded in 1% (w/v) agarose gel were used. PSFs were calculated by analyzing the z-stack images of the beads using PSFj software ver.3  [The sample chamber was filled with 10 μg/mL fluorescein solution to measure beam line profiles. In order to evaluate the beam extent and thickness, FWHMs along the longitudinal (er 2018) .w/v)%) containing methylene blue. Dechorionation of eggs was performed using a hatching enzyme before microscopy observations. The transgenic strain FLT4-EGFP, which expresses EGFP [Medaka fish were maintained in freshwater tanks with a water circulating system  at 26–28 °C under the conditions of a 14-h-light and 10-h-dark cycle (08:30–22:30 light). Fish were fed artemia larvae and a powdered diet daily. The spawned eggs were collected in the morning, and eggs were incubated at 28 °C in a dish filled with diluted artificial seawater (0.03 (ses EGFP  in lymphw/v) agarose gel set in the sample holder, which was put into the sample chamber filled with the embryonic culture medium [Before the observation, embryos were dechorionated using the hatching enzyme, which was provided by National BioResource Project (NBRP) Medaka. For observations, we embedded embryo samples into a 1% .Data are presented as box plots, with the lines of the box indicating the first quantile, median, and third quantile. Upper and lower whiskers indicate the minimum and maximum, respectively. The cross mark represents the average. The asterisk indicates a